Cardamonin inhibits the progression of oesophageal cancer by inhibiting the PI3K/AKT signalling pathway

Abstract

Background: Oesophageal cancer is the most common malignant tumour with a poor prognosis, and the current treatment methods are limited. Therefore, identifying effective treatment methods has become a research hotspot. Cardamonin (CAR) is a natural chalcone compound and has been reported to play an anticancer role in several cancers. However, its function in oesophageal cancer and the possible underlying mechanism are still unclear. The purpose of this study was to demonstrate the anticancer effect of CAR on oesophageal cancer in vivo and in vitro and to explore the underlying mechanism. Materials and Methods: MTT, crystal violet, and colony formation assays were used to detect oesophageal cancer cell proliferation. The effects of CAR on oesophageal cancer cell migration and invasion were detected by wound healing assay and Transwell assay. Hoechst 33258 staining and flow cytometry were used to detect cell apoptosis. Protein expression levels were detected by Western blot. A tumour xenograft model was established to further test the effect of CAR on the growth of oesophageal cancer in vivo. Results: The results showed that CAR inhibited the proliferation, migration, and invasion of oesophageal cancer cells in a concentration-dependent manner and induced apoptosis. Furthermore, the Western blot assay showed that CAR could suppress metastasis by inhibiting epithelial-mesenchymal transition (EMT) as indicated by downregulated expression of the mesenchymal markers N-cadherin and vimentin, the EMT transcription factor Snail, and matrix metalloproteinases (MMPs) and upregulated expression of the epithelial marker E-cadherin. CAR was associated with upregulation of the pro-apoptotic proteins Bax and Bad and downregulation of the anti-apoptotic protein Bcl-2 and triggered the mitochondrial apoptosis pathway, which in turn promoted caspase-3 activation and subsequent cleavage of PARP; however, the mitochondria-related apoptotic effects induced by CAR were blocked by caspase inhibitor Z-VAD-FMK pretreatment, which prevented programmed cell death triggered by CAR. In addition, CAR reduced the phosphorylation level of downstream effector molecules of phosphatidylinositol 3 kinase (PI3K) in a dose-dependent manner, and treatment with the PI3K agonist 740Y-P could partially reverse the anticancer effect of CAR, demonstrating that CAR played an antitumour role by inhibiting the PI3K/AKT signalling pathway in oesophageal cancer cells. Moreover, the EC9706 xenograft model further confirmed that CAR can significantly inhibit tumour growth in vivo. Conclusion: In summary, CAR exhibited a strong anticancer effect on human oesophageal cancer cells and promoted apoptosis by inhibiting the PI3K/AKT signalling pathway, suggesting that CAR can be used as new strategy for oesophageal cancer treatment.

Keywords: PI3K/AKT signalling pathway; antitumour; cardamonin; growth; oesophageal cancer.

Figure 1

CAR inhibited oesophageal cancer cell proliferation. (A and B) EC9706 and TE10 cells were treated with various dosages of CAR, and the cell growth rate was examined by MTT assay; with increases in the drug concentration and treatment time, the growth of EC9706 and TE10 cells was obviously inhibited. The cells were treated with the screened CAR concentration, and (C and D) MTT assay and (E-H) crystal violet staining results further verified that CAR inhibited the growth of oesophageal cancer cell lines EC9706 and TE10 in concentration- and time-dependent manners. (I-L) Colony formation assay showed that CAR inhibited the growth of oesophageal cancer cell lines EC9706 and TE10 in a concentration-dependent manner. (M and N) Western blot assay results showed that PCNA protein expression in EC9706 cells decreased with increasing CAR concentrations with β-actin as the loading control. All the above quantitative results are presented as the mean ± SD (n = 3, each group). *p < 0.05, **p < 0.01, ***p < 0.001 vs the NC group.

Figure 2

CAR induced oesophageal cancer cell apoptosis. (A-D) EC9706 and TE10 cells were treated with different concentrations of CAR or DMSO for 48 h and then stained with Hoechst 33258. Apoptotic cells with dense chromatin were statistically analysed, and the apoptosis rate was quantified. (E-H) Flow cytometry was used to detect the effect of CAR on oesophageal cancer cell apoptosis. The results proved that CAR induced apoptosis in a concentration-dependent manner. (I and J) The expression levels of apoptosis-related proteins were detected by Western blot with β-actin as the loading control, and with increasing CAR concentrations, the expression levels of cleaved caspase-3, cleaved PARP, Bax, and Bad were upregulated, while the expression levels of caspase-3, PARP, and Bcl2 were downregulated. All the above quantitative results are presented as the mean ± SD (n = 3, each group). *p < 0.05, **p < 0.01, ***p < 0.001 vs the NC group.

Figure 3

CAR inhibited oesophageal cancer cell migration and invasion. (A-D) A wound-healing assay showed the migratory abilities of EC9706 and TE10 cells treated with different concentrations of CAR or DMSO; the statistical results for cell migration width at 0 h and 24 h are shown. (E-H) A representative image of EC9706 and TE10 cells treated with different concentrations of CAR or DMSO in a transwell migration assay; migrating cells were counted under five randomly selected visual fields. (I-L) A representative image of EC9706 and TE10 cells treated with different concentrations of CAR or DMSO in a Transwell Matrigel invasion assay; invading cells were counted under five randomly selected visual fields. The above results proved that CAR inhibited the migration and invasion of oesophageal cancer cell lines EC9706 and TE10 in a concentration-dependent manner. (M and N) The expression levels of EMT-related marker proteins in EC9706 cells treated with different concentrations of CAR or DMSO were detected by Western blot with β-actin as the loading control.With increasing CAR concentrations, E-cadherin expression was upregulated, while the expression levels of vimentin, N-cadherin, EMT transcription factor Snail, MMP2, MMP7, and MMP9 were downregulated. All the above quantitative results are presented as the mean ± SD (n = 3, each group). *p < 0.05, **p < 0.01, ***p < 0.001 vs NC group.

Figure 4

CAR inhibited the PI3K/AKT signalling pathway. (A and B) The protein levels of PI3K, p-PI3K and its downstream effector molecule AKT, and p-AKT were examined by Western blot in EC9706 cells treated with different concentrations of CAR or DMSO, and β-actin served as the loading control. With increasing CAR concentrations, the protein levels of p-PI3K and p-AKT decreased significantly, while the expression levels of PI3K and AKT remained unchanged. The data are presented as the mean ± SD (n = 3, each group). *p < 0.05, **p < 0.01 vs the NC group.

Figure 5

740Y-P reversed the inhibitory effect of CAR on oesophageal cancer cells. (A and B) The oesophageal cancer cell line EC9706 was incubated with CAR and the PI3K agonist 740Y-P or subjected to co-incubation, and Western blot results proved that 740Y-P could partially reverse the inhibitory effect of CAR on the PI3K/AKT pathway. Similarly, EC9706 and TE10 cells were treated with CAR and the PI3K agonist 740Y-P or with co-incubation. (C and D) MTT assay and (E and F) colony formation assay results confirmed that the PI3K agonist 740Y-P could partially reverse the antiproliferation effect of CAR. (G and H) Scratch healing assay, (I and J) Transwell migration assay, and (K and L) Transwell invasion assay results showed that the PI3K agonist 740Y-P can partially reverse the anti-migration and anti-invasion effects of CAR on oesophageal cancer cells. (M and N) Hoechst 33258 staining and (O and P) flow cytometry were used to verify the effect of CAR and 740Y-P or co-incubation on oesophageal cancer cell apoptosis, and the results showed that 740Y-P could partially reverse the apoptosis-promoting effect of CAR on EC9706 and TE10 cells. (Q-V) Western blot and quantitative analysis were used to detect the protein expression levels of Bcl2, Bax, Bad, caspase-3, cleaved caspase-3, MMP2, MMP9, N-cadherin, E-cadherin, Snail, vimentin, and PCNA after EC9706 cells were treated with 740Y-P and CAR or with co-incubation. The results showed that 740Y-P could partially reverse the expression of apoptosis-related proteins, EMT-related proteins, and PCNA. The data are expressed as the mean ± SD (n = 3, each group). Compared with the NC group, *p < 0.05, **p < 0.01, ***p < 0.001; Compared with the 740Y-P group, #p < 0.05, ## p < 0.01, ### p < 0.001; Compared with the Cardamonin group, & p < 0.05, && p < 0.01, &&& p < 0.001.

Figure 6

CAR inhibited the growth of EC9706 cells in vivo. (A and B) Nude mice were perfused with different concentrations of CAR, and tumour size was measured every three days. Compared with the control group, the CAR treatment group showed significantly inhibited tumour growth. (C) The weights of mice were measured every three days. Compared with that in the control group, weight in the CAR treatment group did not significantly change. (D and E) PCNA, p-PI3K, p-AKT, Bcl2, and vimentin were detected by immunohistochemistry, and with increasing CAR concentrations, the expression levels of PCNA, p-PI3K, p-AKT, Bcl2, and vimentin were downregulated. The quantitative data are presented as the mean ± SD (n = 3 in each group). *p < 0.05, **p < 0.01, ***p < 0.001 vs the NC group.

Figure 7

An illustration showing how CAR exerts anticancer activity in oesophageal cancer cells by suppressing the PI3K/AKT signalling pathway.